Biology subjects

Mihailescu, M. R.

Publications and source records attributed to Mihailescu, M. R..

5 recordsLinked to original sources

bioRxiv2026

The role of a G-quadruplex structure in mediating the interactions of miR-1249-3p with the 3-UTR of the HNRNPK mRNA

Heterogenous nuclear riboprotein K (hnRNP K) is an important regulator of multiple signaling pathways involved in DNA repair and function whose expression has been found to be mis-regulated in many cancers and neurological diseases. The expression regulation of hnRNP K is not fully understood however it has been shown that the microRNA miR-1249-3p targets its mRNA 3-untranslated region (UTR), regulating its translation. In this study we identified a guanosine-rich sequence in the HNRNPK 3-UTR within the miR-1249-3p binding site that has the potential to form a G-quadruplex (GQ) structure. By using various biophysical methods, we show that this sequence forms a parallel GQ structure which modulates the binding of miR-1249-3p to HNRNPK 3-UTR in vitro. Additionally, using luciferase reporter assays, we determined that this GQ structure affects the hnRNP K protein expression, suggesting modulation of the binding interaction of miR-1249-3p to the 3-UTR of HNRNPK. This HNRNPK mRNA GQ-miR binding axis is an unexplored mechanism of regulation, offering a novel therapeutic target for diseases caused by abnormal hnRNP K expression.

biochemistry↗

bioRxiv2025

G-Quadruplex and i-Motif Structures in the SHMT1 5UTR Modulate Gene Expression

Multiple sclerosis (MS) is a fatal neurodegenerative disease that progresses by eroding the myelin sheath and exposing the neuron, leading to neuronal degradation and death. While MS remains without an effective treatment or cure, studies have identified genes that are dysregulated in MS patients and predicted to be involved with disease progression. These genes are primarily involved in controlling DNA methylation: a process required for regulating gene expression, which is critical for cellular health. Having identified potential genetic risk factors, research is focused on how to manipulate the expression of these genes by offsetting DNA methylation errors in patients through the targeting of DNA and RNA secondary structure formation. Serine hydroxy methyltransferase 1 (SHMT1), a key player in DNA methylation, was determined to be upregulated in MS patients. Here, we identified and characterized a hybrid 3+1 G-quadruplex (GQ) and i-motif (iM) structures in the SHMT1 DNA 5 untranslated region and a parallel GQ in the corresponding mRNA. Additionally, we found that the GQ/iM structures suppress the mRNA levels and protein expression of a reporter gene. Together, these data suggest that GQ/iM structures are necessary for SHMT1 regulation, which could serve as a target for therapeutic intervention for MS patients.

biochemistry↗

bioRxiv2023

Host microRNA interactions with the SARS-CoV-2 viral genome 3'-untranslated region

The 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has marked the spread of a novel human coronavirus. While the viral life cycle is well understood, most of the interactions at the virus-host interface remain elusive. Furthermore, the molecular mechanisms behind disease severity and immune evasion are still largely unknown. Conserved elements of the viral genome such as secondary structures within the 5- and 3-untranslated regions (UTRs) serve as attractive targets of interest and could prove crucial in furthering our understanding of virus-host interactions. It has been proposed that microRNA (miR) interactions with viral components could be used by both the virus and host for their own benefit. Analysis of the SARS-CoV-2 viral genome 3-UTR has revealed the potential for host cellular miR binding sites, providing sites for specific interactions with the virus. In this study, we demonstrate that the SARS-CoV-2 genome 3-UTR binds the host cellular miRNAs miR-760-3p, miR-34a-5p, and miR-34b-5p, which have been shown to influence translation of interleukin-6 (IL-6), the IL-6 receptor (IL-6R), as well as progranulin (PGRN), respectively, proteins that have roles in the host immune response and inflammatory pathways. Furthermore, recent work suggests the potential of miR-34a-5p and miR-34b-5p to target and inhibit translation of viral proteins. Native gel electrophoresis and steady-state fluorescence spectroscopy were utilized to characterize the binding of these miRs to their predicted sites within the SARS-CoV-2 genome 3-UTR. Additionally, we investigated 2-fluoro-D-arabinonucleic acid (FANA) analogs of these miRNAs as competitive binding inhibitors for these miR binding interactions. The mechanisms detailed in this study have the potential to drive the development of antiviral treatments for SARS-CoV-2 infection, and provide a potential molecular basis for cytokine release syndrome and immune evasion which could implicate the host-virus interface. Author SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now plagued the world for over three years. In this time, scientific advancements have allowed for the development of mRNA vaccines and targeted antiviral drugs. However, many mechanisms of the viral life cycle, as well as the interactions at the host-virus interface, remain unknown. The host immune response is of particular interest in combating SARS-CoV-2 infection, with observed dysregulation in both severe and mild cases of infection. To uncover the link between SARS-CoV-2 infection and observed immune dysregulation, we investigated host microRNAs associated with the immune response, particularly miR-760-3p, miR-34a-5p, and miR-34b-5p and emphasize them as targets of binding by the viral genome 3-UTR. We utilized biophysical methods to characterize the interactions between these miRs and the SARS-CoV-2 viral genome 3-UTR. Lastly, we introduce 2-fluoro-D-arabinonucleic acid analogs of these microRNAs as disruptors of the binding interactions, with intent of therapeutic intervention.

biochemistry↗

bioRxiv2023

Effect of the SARS-CoV-2 Delta-associated G15U mutation on the s2m element dimerization and its interactions with miR-1307-3p

The stem loop 2 motif (s2m), a highly conserved 41-nucleotide hairpin structure in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, serves as an attractive therapeutic target that may have important roles in the virus life cycle or interactions with the host. However, the conserved s2m in Delta SARS-CoV-2, a previously dominant variant characterized by high infectivity and disease severity, has received relatively less attention than that of the original SARS-CoV-2 virus. The focus of this work is to identify and define the s2m changes between Delta and SARS-CoV-2 and subsequent impact of those changes upon the s2m dimerization and interactions with the host microRNA miR-1307-3p. Bioinformatics analysis of the GISAID database targeting the s2m element reveals a greater than 99% correlation of a single nucleotide mutation at the 15th position (G15U) in Delta SARS-CoV-2. Based on 1H NMR assignments comparing the imino proton resonance region of s2m and the G15U at 19{degrees}C, we find that the U15-A29 base pair closes resulting in a stabilization of the upper stem without overall secondary structure deviation. Increased stability of the upper stem did not affect the chaperone activity of the viral N protein, as it was still able to convert the kissing dimers formed by s2m G15U into a stable duplex conformation, consistent with the s2m reference. However, we find that the s2m G15U mutation drastically reduces the binding affinity of the host miR-1307-3p. These findings demonstrate that the observed G15U mutation alters the secondary structure of s2m with subsequent impact on viral binding of host miR-1307-3p, with potential consequences on the immune response.

biochemistry↗

bioRxiv2020

SARS-CoV-2 highly conserved s2m element dimerizes via a kissing complex and interacts with host miRNA-1307-3p

The ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the 3 UTR is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the s2m element of SARS-CoV-2 dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular miRNA-1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19 and other coronavirus-related diseases.

biochemistry↗